The present invention is in the field of recombinant DNA technology. This invention is directed to a gene sequence and a protein that effects the ability of cells to become senescent.
Normal human diploid cells have a finite potential for proliferative growth (Hayflick, L. et al., Exp. Cell Res. 25:585 (1961); Hayflick, L., Exp. Cell Res. 37: 614 (1965)). Indeed, under controlled conditions in vitro cultured human cells can maximally proliferate only to about 80 cumulative population doublings. The proliferative potential of such cells has been found to be a function of the number of cumulative population doublings which the cell has undergone (Hayflick, L. et al., Exp. Cell Res. 25: 585 (1961); Hayflick, L. et al., Exp. Cell Res. 37: 614 (1985)). This potential is also inversely proportional to the in vivo age of the cell donor (Martin, G. M. et al., Lab. Invest. 23:86 (1979); Goldstein, S. et al., Proc. Natl. Acad. Sci. (U.S.A.) 64:155 (1969); Schneider, E. L., Proc. Nati. Acad. Sci. (U.S.A.) 73:3584 (1976); LeGuilty, Y. et al., Gereontologia 19:303 (1973)).
Cells that have exhausted their potential for proliferative growth are said to have undergone xe2x80x9csenescence.xe2x80x9d Cellular senescence in vitro is exhibited by morphological changes and is accompanied by the failure of a cell to respond to exogenous growth factors. Cellular senescence, thus, represents a loss of the proliferative potential of the cell. Although a variety of theories have been proposed to explain the phenomenon of cellular senescence in vitro, experimental evidence suggests that the age-dependent loss of proliferative: potential may be the function of a genetic program (Orgel, L. E., Proc. Natl. Acad. Sci. (U.S.A.) 49:517 (1963); De Mars, R. et al., Human Genet. 16:87 (1972); M. Buchwald, Mutat. Res. 44:401 (1977); Martin, G. M. et al., Amer. J. Pathol. 74:137 (1974); Smith, J. R. et al., Mech. Age. Dev. 13:387 (1980): Kirkwood, T. B. L. et al., Theor. Biol. 53:481 (1975).
Cell fusion studies with human fibroblasts in vitro have demonstrated that the quiescent phenotype of cellular senescence is dominant over the proliferative phenotype (Pereira-Smith, O. M et al., Somat. Cell Genet. 6:731 (1982); Norwood, T. H. et al., Proc. Nati. Acad. Sci. (U.S.A.) 71:223 (1974); Stein, G. H. et al., Exp. Cell Res. 130:155 (1979)).
Insight into the phenomenon of senescence has been gained from studies in which senescent and young (i.e. non-senescent) cells have been fused to form heterodikaryons. In order to induce senescence in the xe2x80x9cyoungxe2x80x9d nucleus of the heterodikaryon (as determined by an inhibition in the synthesis of DNA), protein synthesis must occur in the senescent cell prior to fusion (Burmer, G. C. et al., J. Cell. Biol. 94:187 (1982); Drescher-Lincoln, C. K. et al., Exp. Cell Res. 144:455 (1983); Burner, G. C. et al., Exp. Cell Res. 145:708 (1983); Drescher-Lincoln, C. K. et al., Exp. Cell Res. 153:208 (1984).
Likewise, microinjection of senescent fibroblast mRNA into young fibroblasts has been found to inhibit the ability of the young cells to synthesize DNA (Lumpkin, C. K. et al., Science 232:393 (1986)). Researchers have identified unique mRNAs that are amplified in senescent cells in vitro (West, M. D. et al., Exp. Cell Res. 184:138 (1989); Giordano, T. et al., Exp. Cell Res. 185:399 (1989)).
The human diploid endothelial cell presents an alternative cell type for the study of cellular senescence because such cells mimic cellular senescence in vitro (Maciag, T. et al., J. Cell. Biol. 91:420 (1981); Gordon, P. B. et al., In Vitro 19:661 (1983); Johnson, A. et al., Mech Age. Dev. 18:1 (1982); Thornton, S. C. et al., Science 222:623 (1983); Van Hinsbergh, V. W. M. et al., Eur. J. Cell Biol. 42:101 (1986); Nichols, W. W. et al., J. Cell. Physiol. 132:453 (1987)).
In addition, the human endothelial cell is capable of expressing a variety of functional and reversible phenotypes. The endothelial cell exhibits several quiescent and non-terminal differentiation phenotypes (Folkman, J. et al., Nature 288:551 (1980); Maciag, T. et al., J. Cell Biol. 94:511 (1982); Madri. J. A. et al., J. Cell Biol. 97:153 (1983); Montesano, R., J. Cell Biol. 99:1706 (1984); Montesano, R. et al., J. Cell Physiol. 34:460 (1988)).
It has been suggested that the pathway of human cell differentiation in vitro involves the induction of cellular quiescence mediated by cytokines that inhibit growth factor-induced endothelial cell proliferation in vitro (Jay, M. et al., Science 228:882 (1985); Madri, J. A. et al., In Vitro 23:387 (1987); Kubota, Y. et al., J. Cell Biol. 107:1589 (1988); Ingber, D. E. et al., J. Cell Biol. 107:317 (1989)).
Inhibitors of endothelial cell proliferation also function as regulators of immediate-early transcriptional events induced during the endothelial cell differentiation in vitro, which involves formation of the capillary-like, tubular endothelial cell phenotype (Maciag, T., In: Imp. Adv. Oncol. (De Vita, V. T. et al., eds., J. B. Lippincott. Philadelphia, 42 (1990); Goldgaber, D. et al., Proc. Natl. Acad. Sci. (U.S.A.) 86:7606 (1990); Hla, T. et al., Biochem. Biophys. Res. Commun. 167:637 (1990)). The inhibitors of cell proliferation include:
1. Interleukin-1a (IL-1a) (Montesano, R. et al., J. Cell Biol. 99:1706 (1984); Montesano, R. et al., J. Cell Physiol. 122:424 (1985); Maciag, T. et al. (Science 249:1570-1574 (1990));
2. Tumor necrosis factor (Frater-Schroder, M. et al., Proc. Natl. Acad. Sci. (U.S.A.) 84:5277 (1987); Sato, N. et al., J. Natl. Cancer Inst. 76:1113 (1986); Pber, J. P., Amer. J. Pathol. 133:426 (1988); Shimada, Y. et al., J. Cell Physiol. 142:31 (1990));
3. Transforming growth factor-xcex2 (Baird, A. et al., Biochem. Biophys. Res. Commun. 138:476 (1986); Mullew, G. et al., Proc. Natl. Acad. Sci. (U.S.A.) 84:5600 (1987); Mairi, J. A. et al., J. Cell Biol. 106:1375 (1988));
4. Gamma-interferon (Friesel, R. et al., J. Cell Biol. 104:689 (1987); Tsuruoka, N. et al., Biochem. Biophys. Res. Commun. 155:429 (1988)) and
5. The tumor promoter, phorbol myristic acid (PMA) (Montesano, R. et al., Cell 42:469 (1985); Doctrow, S. R. et al., J. Cell Biol. 104:679 (1987); Montesano, R. et al., J. Cell. Physiol. 130:284 (1987); Hoshi, H. et al., FASAB J. 2:2797 (1988)).
The prospect of reversing senescence and restoring the proliferative potential of cells has implications in many fields of endeavor. Many of the diseases of old age are associated with the loss of this potential. Restoration of this ability would have far-reaching implications for the treatment of this disease, of other age-related disorders, and, of aging per se.
In addition, the restoration of proliferative potential of cultured cells has uses in medicine and in the pharmaceutical industry. The ability to immortalize nontransformed cells can be used to generate an endless supply of certain tissues and also of cellular products.
The significance of cellular senescence has accordingly been appreciated for several years (Smith, J. R., Cellular Ageing, In: Monographs in Developmental Biology; Sauer, H. W. (Ed.), S. Karger, New York, N.Y. 17:193-208 (1984); Smith, J. R. et al. Exper. Gerontol. 24:377-381 (1989), herein incorporated by reference). Researchers have attempted to clone genes relevant to cellular senescence. A correlation between the existence of an inhibitor of DNA synthesis and the phenomenon of cellular senescence has been recognized (Spiering, A. I. et al., Exper. Cell Res. 179:159-167 (1988); Pereira-Smith, O. M. et al., Exper. Cell Res. 160:297-306 (1985); Drescher-Lincoln, C. K. et al., Exper. Cell Res. 153:208-217 (1984); Drescher-Lincoln, C. K. et al, Exper. Cell Res. 144:455-462 (1983)). Moreover, the relative abundance of certain senescence-associated RNA molecules has been identified (Lumpkin, C. K. et al, Science 232:393-395 (1986)).
Several laboratories have used the xe2x80x9csubtraction-differentialxe2x80x9d screening method to identify cDNA molecules derived from RNA species that are preferentially present in senescent cells (Kleinsek, D. A., Age 12:55-60 (1989); Giordano, T. et at., Exper. Cell. Res. 185:399-406 (1989); Sierra, F. et al., Molec. Cell. Biol. 9:5610-5616 (1989); Pereira-Smith, O. M. et al., J Cell. Biochem. (Suppl 0 (12 part A)). 193 (1988); Kleinsek, D. A., Smith, J. R., Age 10:125 (1987)).
In one method, termed xe2x80x9csubtraction-differentialxe2x80x9d screening, a pool of cDNA molecules is created from senescent cells, and then hybridized to cDNA or RNA of growing cells in order to xe2x80x9csubtract outxe2x80x9d those cDNA molecules that are complementary to nucleic acid molecules, present in growing cells. Although useful, for certain purposes, the xe2x80x9csubtraction-differentialxe2x80x9d method suffers from the fact that it is not possible to determine whether a senescence-associated cDNA molecule is associated with the cause of senescence, or is produced as a result of senescence. Indeed, many of the sequences identified in this manner have been found to encode proteins of the extra-cellular matrix. Changes in the expression of such proteins would be unlikely to cause senescence.
This application is a continuation-in-part of PCT US94/09700 (filed Aug. 26, 1994), herein incorporated by reference in its entirety. The present invention concerns, in part, the observation that normal human cells exhibit a limited replicative potential in vitro and become senescent after a certain number of divisions. As the cells become senescent, they show several morphological and biochemical changes, such as enlargement of cell size, changes of extracellular matrix components, unresponsiveness: to mitogen stimulation and failure to express growth regulated genes.
The present invention identifies an inhibitor of DNA synthesis that is produced in senescent cells. This inhibitor plays a crucial role in the expression of the senescent phenotype. The gene coding for the inhibitor was identified by incorporating a senescent cell cDNA library into a mammalian expression vector. The cDNA library was then transfected into young, cycling cells to identify those library members that suppressed the initiation of DNA synthesis.
Efficient DEAE dextran-mediated transfection enabled the isolation of putative senescent cell derived inhibitor (SDI) sequences in three distinct cDNA clones. The expression of one (SDI-1) increased 20 fold at cellular senescence, whereas that of the others (SDI-2 and SDI-3) remained constant.
In summary, the present invention achieves the cloning of an inhibitor of DNA synthesis using a functional assay. This method may be applied to clone other genes involved in negative regulation of the cell cycle, such as tissue specific differentiation and tumor suppression genes. Using this method, three inhibitor sequences have been cloned. One of these sequences (SDI-1) appears to be closely related to cellular senescence.
In detail, the invention provides a nucleic acid molecule that encodes a protein or polypeptide capable of inhibiting DNA synthesis in a recipient cell.
In particular, the invention provides a liposome preparation that comprises an SDI molecule, and particularly one that comprises:
(a) a mixture of a polycationic and a neutral lipid; and
(b) an SDI molecule selected from the group consisting of SDI-1 protein and an SDI-1-encoding nucleic acid molecule.
The invention particularly provides such liposome preparations wherein the polycationic lipid is 2,3-dioleyloxy-N-[2(sperminecarboxamido)-ethyl]-N,N-dimethyl-1-propanaminium-trifluoroacetate (DOSPA) and/or wherein the neutral lipid is dioleolyphosphatidylethanolamine (DOPE).
The invention particularly concerns the embodiments wherein (A) the SDI molecule is an SDI-1-encoding nucleic acid molecule that is operably linked to a promoter, but is separated from the operably linked promoter by a non-translated intervening polynucleotide and/or (B) wherein the SDI-1-encoding nucleic acid molecule is operably linked to a promoter, and contains a non-translated intervening polynucleotide which separates a region of the SDI-1-encoding nucleic acid that encodes part of SDI-1 from a region of the SDI-1-encoding nucleic acid that encodes a different part of SDI-1.
The invention also provides a method for preparing a liposome preparation of SDI molecules which comprises incubating liposomes that comprise a mixture of a polycationic and a neutral lipid with an SDI molecule selected from the group consisting of SDI-1 protein and an SDI-1-encoding nucleic acid molecule.
The invention also concerns a method of providing an SDI molecule to a cell which comprises:
(A) contacting the cell with a liposome preparation that comprises a mixture of a polycationic and a neutral lipid and an SDI molecule selected from the group consisting of SDI-1 protein and an SDI-1-encoding nucleic acid molecule; and
(B) permitting the intracellular delivery of the SDI-1 molecule of the liposome preparation.
The invention also concerns an SDI-1-encoding nucleic acid molecule, operably linked to a promoter, but separated from the operably linked promoter by a non-translated intervening polynucleotide.
The invention also provides a method of transcribing an SDI-1-encoding nucleic acid molecule which comprises:
(A) providing to a cell the SDI-1-encoding nucleic acid molecule, operably linked to a promoter, but separated from the operably linked promoter by a non-translated intervening polynucleotide; and
(B) permitting the promoter to mediate the transcription of the SDI-1-encoding nucleic acid molecule.
The invention also provides a nucleic acid molecule that encodes SDI-1, a fragment of SDI-1, an SDI-1 fusion protein or a mimetic or analog of SDI-1.
The invention also provides a protein or polypeptide capable of inhibiting DNA synthesis in a recipient cell, wherein the protein or polypeptide is SDI-1, a fragment of SDI-1, an SDI-1 fusion protein or a mimetic or analog of SDI-1.
The invention additionally provides a method for treating a disease of undesired cellular proliferation which comprises providing to a recipient a nucleic acid molecule that encodes SDI-1, a fragment of SDI-1, an SDI-1 fusion protein, an SDI-1 mimetic, or an analog of SDI-1.
The invention additionally provides a method for treating a disease of undesired cellular proliferation which comprises providing to a recipient a protein or polypeptide capable of inhibiting DNA synthesis in a recipient cell, wherein the protein or polypeptide is SDI-1, a fragment of SDI-1, an SDI-1 fusion protein or a mimetic or analog of SDI-1.
The invention additionally provides a method for treating a disease of undesired cellular quiescence which comprises providing to a recipient a nucleic acid molecule that encodes an inhibitor of SDI-1.
The invention additionally provides a method for treating a disease of undesired cellular quiescence which comprises providing to a recipient a protein, polypeptide, or organic molecule capable of inhibiting SDI-1.